Microbicidal composition

ABSTRACT

A synergistic microbicidal composition having two components. The first component is a nonionic surfactant with structure: R 2 O(CH 2 CH(CH 3 )O) 3 (CH 2 CH 2 O) 7 H, where R 2  is a mixture of C 8 -C 14  linear alkyl groups. The second component is tris(hydroxymethyl)nitromethane. The weight ratio of the nonionic surfactant to tris(hydroxymethyl)nitromethane is from 1:0.16 to 1:1.8286.

This invention relates to microbicidal compositions containingcis-1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride (CTAC),4,4-dimethyloxazolidine (DMO) or tris(hydroxymethyl)nitromethane (TrisNitro) and a surfactant.

A composition containing 5-chloro-2-methylisothiazolin-3-one,2-methylisothiazolin-3-one and a nonionic dispersant is disclosed inU.S. Pat. No. 4,295,932. The composition contains a 3:1 mixture of5-chloro-2-methylisothiazolin-3-one and 2-methylisothiazolin-3-one, anda copolymer of ethylene oxide and propylene oxide which appears to havethe same composition as PLURONIC L61 or TERGITOL L61 dispersant.However, there is a need for combinations of microbicides havingsynergistic activity against various strains of microorganisms toprovide effective control of the microorganisms. Moreover, there is aneed for such combinations containing lower levels of individualmicrobicides for environmental and economic benefit. The problemaddressed by this invention is to provide such synergistic combinationsof microbicides.

STATEMENT OF THE INVENTION

The present invention is directed to a synergistic microbicidalcomposition comprising: (a) a nonionic surfactant with structure:

R¹O(CH₂CH(CH₃)O)₅(CH₂CH₂O)₉H

where R¹ is a C₈ alkyl group; and (b)cis-1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride; whereina weight ratio of said nonionic surfactant tocis-1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride is from1:0.2 to 1:1.1429.

The present invention is further directed to a synergistic microbicidalcomposition comprising: (a) a nonionic surfactant with structure:

R²O(CH₂CH(CH₃)O)₃(CH₂CH₂O)₅H

where R² is a mixture of C₈-C₁₄ linear alkyl groups; and (b)cis-1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride; whereina weight ratio of said nonionic surfactant tocis-1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride is from1:0.04 to 1:0.9143.

The present invention is further directed to a synergistic microbicidalcomposition comprising: (a) a nonionic surfactant with structure:

R²O(CH₂CH(CH₃)O)₃(CH₂CH₂O)₇H

where R² is a mixture of C₈-C₁₄ linear alkyl groups; and (b)cis-1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride; whereina weight ratio of said nonionic surfactant tocis-1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride is from1:0.016 to 1:0.0571.

The present invention is further directed to a synergistic microbicidalcomposition comprising: (a) a nonionic surfactant with structure:

R¹O(CH₂CH(CH₃)O)₅(CH₂CH₂O)₉H

where R¹ is a C₈ alkyl group; and (b) 4,4-dimethyloxazolidine; wherein aweight ratio of said nonionic surfactant to 4,4-dimethyloxazolidine isfrom 1:0.0286 to 1:0.1143 or 1:0.32 to 1:0.9143.

The present invention is further directed to a synergistic microbicidalcomposition comprising: (a) a nonionic surfactant with structure:

R²O(CH₂CH(CH₃)O)₃(CH₂CH₂O)₅H

where R² is a mixture of C₈-C₁₄ linear alkyl groups; and (b)4,4-dimethyloxazolidine; wherein a weight ratio of said nonionicsurfactant to 4,4-dimethyloxazolidine is from 1:0.06 to 1:0.6857.

The present invention is further directed to a synergistic microbicidalcomposition comprising: (a) a nonionic surfactant with structure:

R²O(CH₂CH(CH₃)O)₃(CH₂CH₂O)₇H

where R² is a mixture of C₈-C₁₄ linear alkyl groups; and (b)4,4-dimethyloxazolidine; wherein a weight ratio of said nonionicsurfactant to 4,4-dimethyloxazolidine is from 1:0.024 to 1:0.2743.

The present invention is further directed to a synergistic microbicidalcomposition comprising: (a) a nonionic surfactant with structure:

R²O(CH₂CH(CH₃)O)₃(CH₂CH₂O)₇H

where R² is a mixture of C₈-C₁₄ linear alkyl groups; and (b)tris(hydroxymethyl)nitromethane; wherein a weight ratio of said nonionicsurfactant to tris(hydroxymethyl)nitromethane is from 1:0.16 to1:1.8286.

The present invention is further directed to methods for inhibiting thegrowth of microorganisms in aqueous media by adding to an aqueous mediuma nonionic surfactant as described herein andcis-1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride,4,4-dimethyloxazolidne or tris(hydroxymethyl)nitromethane in the ratiosdescribed herein.

DETAILED DESCRIPTION OF THE INVENTION

“CTAC” is cis-1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantanechloride, CAS No. 51229-78-8, “DMO” is 4,4-dimethyloxazolidine, CAS No.51200-87-4, and “Tris Nitro” is tris(hydroxymethyl)nitromethane, CAS No.126-11-4. As used herein, the following terms have the designateddefinitions, unless the context clearly indicates otherwise. The term“microbicide” refers to a compound capable of inhibiting the growth ofor controlling the growth of microorganisms; microbicides includebactericides, fungicides and algaecides. The term “microorganism”includes, for example, fungi (such as yeast and mold), bacteria andalgae. The following abbreviations are used throughout thespecification: ppm=parts per million by weight (weight/weight),mL=milliliter. Unless otherwise specified, temperatures are in degreescentigrade (° C.), references to percentages are percentages by weight(wt %) and amounts and ratios are on an active ingredient basis, i.e.,total weight of CTAC and the nonionic surfactant. Numbers of polymerizedunits of propylene oxide or ethylene oxide are number averages.

Preferably, the weight ratio of the nonionic surfactant with structure:

R¹O(CH₂CH(CH₃)O)₅(CH₂CH₂O)₉H

where R¹ is a C₈ alkyl group to CTAC is from 1:0.2857 to 1:1.1429,preferably from 1:0.5714 to 1:1.1429. Preferably, the weight ratio ofthe nonionic surfactant with structure:

R²O(CH₂CH(CH₃)O)₃(CH₂CH₂O)₅H

where R² is a mixture of C₈-C₁₄ linear alkyl groups to CTAC is from1:0.0457 to 1:0.9143.

The present invention is further directed to a method for inhibiting thegrowth of S. aureus, in an aqueous medium by adding: (a) a nonionicsurfactant with structure:

R²O(CH₂CH(CH₃)O)₃(CH₂CH₂O)₅H

where R² is a mixture of C₈-C₁₄ linear alkyl groups; and (b) CTAC;wherein a weight ratio of said nonionic surfactant to CTAC is from1:0.0457 to 1:0.9143.

The present invention is further directed to a method for inhibiting thegrowth of S. aureus, in an aqueous medium by adding: (a) a nonionicsurfactant with structure:

R²O(CH₂CH(CH₃)O)₃(CH₂CH₂O)₇H

where R² is a mixture of C₈-C₁₄ linear alkyl groups; and (b) Tris Nitro;wherein a weight ratio of said nonionic surfactant to Tris Nitro is from1:0.16 to 1:1.8286.

R² is a mixture of C₈-C₁₄ linear alkyl groups. Preferably, the C₈-C₁₄linear alkyl groups comprise from 50 to 85 wt % C₈-C₁₀ linear alkylgroups and 15 to 50 wt % C₁₂-C₁₄ linear alkyl groups, preferably from 60to 75 wt % C₈-C₁₀ linear alkyl groups and 25 to 40 wt % C₁₂-C₁₄ linearalkyl groups, preferably about 70 wt % C₈-C₁₀ linear alkyl groups andabout 30 wt % C₁₂-C₁₄ linear alkyl groups. Preferably, the linear alkylgroups are derived from seed oil. Preferably, R¹ is 2-ethylhexyl.

Preferably, each of the compositions is substantially free ofmicrobicides other than the nonionic surfactant and CTAC, DMO or TrisNitro, i.e., it has less than 1 wt % of microbicides other than thenonionic surfactant and CTAC, DMO or Tris Nitro based on total weight ofactive ingredients, preferably less than 0.5 wt %, preferably less than0.2 wt %, preferably less than 0.1 wt %. Preferably, when the nonionicsurfactant and CTAC, DMO or Tris Nitro are added to an aqueous medium,the medium is substantially free of other microbicides, i.e., it hasless than 1 wt % of microbicides other than the nonionic surfactant andCTAC, DMO or Tris Nitro based on total weight of active ingredients,preferably less than 0.5 wt %, preferably less than 0.2 wt %, preferablyless than 0.1 wt %.

The compositions of this invention may contain other ingredients, e.g.,defoamers and emulsifiers. The microbicidal compositions of the presentinvention can be used to inhibit the growth of microorganisms or higherforms of aquatic life (such as protozoans, invertebrates, bryozoans,dinoflagellates, crustaceans, mollusks, etc) by introducing amicrobicidally effective amount of the compositions into an aqueousmedium subject to microbial attack. Suitable aqueous media are found in,for example: industrial process water; electrocoat deposition systems;cooling towers; air washers; gas scrubbers; mineral slurries; wastewatertreatment; ornamental fountains; reverse osmosis filtration;ultrafiltration; ballast water; evaporative condensers; heat exchangers;pulp and paper processing fluids and additives; starch; plastics;emulsions; dispersions; paints; latices; coatings, such as varnishes;construction products, such as mastics, caulks, and sealants;construction adhesives, such as ceramic adhesives, carpet backingadhesives, and laminating adhesives; industrial or consumer adhesives;photographic chemicals; printing fluids; household products, such asbathroom and kitchen cleaners; cosmetics; toiletries; shampoos; soaps;personal care products such as wipes, lotions, sunscreen, conditioners,creams, and other leave-on applications; detergents; industrialcleaners; floor polishes; laundry rinse water; metalworking fluids;conveyor lubricants; hydraulic fluids; leather and leather products;textiles; textile products; wood and wood products, such as plywood,chipboard, flakeboard, laminated beams, oriented strandboard, hardboard,and particleboard; petroleum processing fluids; fuel; oilfield fluids,such as injection water, fracture fluids, and drilling muds; agricultureadjuvant preservation; surfactant preservation; medical devices;diagnostic reagent preservation; food preservation, such as plastic orpaper food wrap; food, beverage, and industrial process pasteurizers;toilet bowls; recreational water; pools; and spas.

The specific amount of the microbicidal compositions of this inventionnecessary to inhibit or control the growth of microorganisms in anapplication will vary. Typically, the amount of the composition of thepresent invention is sufficient to control the growth of microorganismsif it provides from 300 to 10,000 ppm (parts per million) activeingredients of the composition. It is preferred that the activeingredients (i.e., nonionic surfactant and CTAC, DMO or Tris Nitro) ofthe composition be present in the medium to be treated in an amount ofat least 500 ppm, preferably at least 600 ppm, preferably at least 800ppm. It is preferred that the active ingredients of the composition bepresent in the locus in an amount of no more than 6,000 ppm, preferablyno more than 5,000 ppm, preferably no more than 4,000 ppm, preferably nomore than 3,000 ppm, preferably no more than 2,500 ppm, preferably nomore than 2,000 ppm. In a method of this invention, a composition istreated to inhibit microbial growth by adding, together or separately,the nonionic surfactant and CTAC, DMO or Tris Nitro, in amounts thatwould produce the concentrations indicated above.

Examples

Surfactants and biocides were evaluated for synergy by determining thesynergy index (S.I.) of the combination. Synergy index was calculatedbased on minimum inhibitory concentrations (MIC) of two antimicrobialcompounds (A and B) alone and in combinations. The tests organisms wereGram negative bacteria (Pseudomonas aeruginosa ATCC #15442), Grampositive bacteria (Staphylococcus aureus ATCC #6538), yeast (Candidaalbicans ATCC#10203) and mold (Aspergillus niger ATCC#16404). Contacttime for the bacteria was 24 and 48 hours, yeast was 48 and 72 hrs, and3 and 7 days for mold. The test was carried out in 96 well microtiterplates.

Surf. A R¹O(CH₂CH(CH₃)O)₅(CH₂CH₂O)₉H, where R¹ is 2-ethylhexylSurf. D R²O(CH₂CH(CH₃)O)₃(CH₂CH₂O)₅HSurf. E R²O(CH₂CH(CH₃)O)₃(CH₂CH₂O)₇HIn Surf. D and Surf. E, R² is a mixture of C₈-C₁₄ linear alkyl groups(70% C₈-C₁₀ linear alkyl and 30% C₁₂-C₁₄ linear alkyl)

Inoculums Used Inoculum Size of organisms (CFU/ml) StaphylococcusPseudomonas Aspergillus Candida aureus aeruginosa niger albicansSurfactants ATCC# 6538 ATCC # 15442 ATCC# 16404 ATCC#10203 Surf. A1.156E+06 8.134E+07 1.156E+06 1.156 + 06 Surf. D 1.808E+05 1.156E+081.156E+06 5.726E+05 Surf. E 1.808E+06 5.727E+07 5.726E+05 1.808E+06

TABLE 4 Media Used Media Used for testing Staphylococcus PseudomonasAspergillus Candida aureus aeruginosa niger albicans ATCC# 6538 ATCC #15442 ATCC# 16404 ATCC#10203 10% Tryptic soy 10% Tryptic soy Potatodextrose Potato dextrose broth broth broth broth

The pH of the Triptic soy broth was 7.3 and the Potato dextrose brothwas 5.1.

The test results for demonstration of synergy of the MIC combinationsare shown in the tables below. Each table shows the results forcombinations of two components against the microorganisms tested withincubation times; the end-point activity in ppm measured by the MIC forcompound A alone (CA), for component B alone (CB), and the mixture (Ca)and (Cb); the calculated SI value; and the range of synergistic ratiosfor each combination tested. SI is calculated as follows:

Ca/CA+Cb/CB=Synergy Index(“SI”)

Wherein:

-   -   CA=concentration of compound A in ppm, acting alone, which        produced an end point (MIC of Compound A).    -   Ca=concentration of compound A in ppm, in the mixture, which        produced an end point.    -   CB=concentration of compound B in ppm, acting alone, which        produced an end point (MIC of Compound B).    -   Cb=concentration of compound B in ppm, in the mixture, which        produced an end point.        When the sum of Ca/CA and Cb/CB is greater than one, antagonism        is indicated. When the sum is equal to one, additivity is        indicated, and when less than one, synergism is demonstrated.

The ratio ranges at which CTAC, DMO or Tris Nitro and the surfactantswere tested are as summarized in the following tables:

CTAC with Surf. A Organism ATCC# From To Ratio Range Staphylococcus 65380.2:20,000 1,000:218.75 1:0.00001-1:4.5714 aureus Aspergillus 164040.2:20,000 1,000:218.75 1:0.00001-1:4.5714 niger Candida 102030.2:20,000 1,000:218.75 1:0.00001-1:4.5714 albicans Pseudomonas 154420.2:20,000 1,000:218.75 1:0.00001-1:4.5714 aeruginosa

CTAC with Surf. E Organism ATCC# From To Ratio Range Staphylococcusaureus 6538 0.2:20,000  1,000:218.75 1:0.00001-1:4.5714 Aspergillusniger 16404 2.0:20,000 10,000:218.75  1:0.0001-1:45.7143 Candidaalbicans 10203 2.0:20,000 10,000:218.75  1:0.0001-1:45.7143 Pseudomonasaeruginosa 15442 0.2:20,000  1,000:218.75 1:0.00001-1:4.5714

CTAC with Surf. D Organism ATCC# From To Ratio Range Staphylococcusaureus 6538 0.2:20,000 1,000:218.75 1:0.00001-1:4.5714 Aspergillus niger16404 0.2:20,000 1,000:218.75 1:0.00001-1:4.5714 Candida albicans 102030.2:20,000 1,000:218.75 1:0.00001-1:4.5714 Pseudomonas aeruginosa 154420.2:20,000 1,000:218.75 1:0.00001-1:4.5714

DMO with Surf. A Organism ATCC# From To Ratio Range Staphylococcusaureus 6538 0.2:20,000 1,000:218.75 1:0.00001-1:4.5714 Aspergillus niger16404 0.2:20,000 1,000:218.75 1:0.00001-1:4.5714 Candida albicans 102030.2:20,000 1,000:218.75 1:0.00001-1:4.5714 Pseudomonas aeruginosa 154420.2:20,000 1,000:218.75 1:0.00001-1:4.5714

DMO with Surf. E Organism ATCC# From To Ratio Range Staphylococcusaureus 6538 0.2:20,000 1,000:218.75 1:0.00001-1:4.5714 Aspergillus niger16404 0.2:20,000 1,000:218.75 1:0.00001-1:4.5714 Candida albicans 102030.2:20,000 1,000:218.75 1:0.00001-1:4.5714 Pseudomonas aeruginosa 154420.2:20,000 1,000:218.75 1:0.00001-1:4.5714

DMO with Surf. D Organism ATCC# From To Ratio Range Staphylococcusaureus 6538 0.2:20,000 1,000:218.75 1:0.00001-1:4.5714 Aspergillus niger16404 0.2:20,000 1,000:218.75 1:0.00001-1:4.5714 Candida albicans 102030.2:20,000 1,000:218.75 1:0.00001-1:4.5714 Pseudomonas aeruginosa 154420.2:20,000 1,000:218.75 1:0.00001-1:4.5714

TrisNitro with Surf. A Organism ATCC# From To Ratio Range Staphylococcusaureus 6538 0.2:20,000 1,000:218.75 1:0.00001-1:4.5714 Aspergillus niger16404 0.02:20,000    100:218.75 1:0.000001-1:0.45714 Candida albicans10203 0.2:20,000 1,000:218.75 1:0.00001-1:4.5714 Pseudomonas aeruginosa15442 0.2:20,000 1,000:218.75 1:0.00001-1:4.5714

TrisNitro with Surf. E Organism ATCC# From To Ratio Range Staphylococcusaureus 6538 0.2:20,000 1,000:218.75 1:0.00001-1:4.5714 Aspergillus niger16404 0.2:20,000 1,000:218.75 1:0.00001-1:4.5714 Candida albicans 102030.2:20,000 1,000:218.75 1:0.00001-1:4.5714 Pseudomonas aeruginosa 154420.2:20,000 1,000:218.75 1:0.00001-1:4.5714

TrisNitro with Surf. D Organism ATCC# From To Ratio Range Staphylococcusaureus 6538 0.2:20,000 1,000:218.75 1:0.00001-1:4.5714 Aspergillus niger16404 0.2:20,000 1,000:218.75 1:0.00001-1:4.5714 Candida albicans 102030.2:20,000 1,000:218.75 1:0.00001-1:4.5714 Pseudomonas aeruginosa 154420.2:20,000 1,000:218.75 1:0.00001-1:4.5714A: Surf. A

B: CTAC Media: PDB

Inoculum size: 1.156E+06

A. niger ATCC#16404 No Synergy C. albicans ATCC#10203 No SynergyA: Surf. A

B: CTAC Media: 1/10 TSB

Inoculum size: 1.156E+06

S. aureus ATCC#6538 No SynergyA: Surf. A

B: CTAC Media: 1/10 TSB

Inoculum size: 1.156E+06

PPM AI MIC Values (3^(rd) Day) Test Alone Combination Ratio Organism CACB Ca Cb S.I. (Ca:Cb) P. aeruginosa >20000 600 20000 500 <1.83 1:0.0250ATCC#15442 >20000 600 10000 500 <1.33 1:0.0500 >20000 600 5000 500 <1.081:0.1000 >20000 600 2500 500 <0.96 1:0.2000 >20000 600 1750 500 <0.921:0.2857 >20000 600 875 500 <0.88 1:0.5714 >20000 600 437.5 500 <0.861:1.1429A: Surf. D

B: CTAC Media: PDB

Inoculum size: 1.156E+06

PPM AI MIC Values (3rd day) Test Alone Combination Ratio Organism CA CBCa Cb S.I. (Ca:Cb) A. niger >20000 1000 10000 600 <1.10 1:0.0600 >200001000 10000 800 <1.30 1:0.0800 ATCC#16404 >20000 1000 5000 800 <1.051:0.1600 >20000 1000 2500 800 <0.93 1:0.3200 >20000 1000 1750 800 <0.891:0.4571 >20000 1000 875 800 <0.84 1:0.9143A: Surf. D

B: CTAC Media: PDB

Inoculum size: 5.726E+05 CFU/ml

PPM AI MIC Values (48 hrs) Test Alone Combination Ratio Organism CA CBCa Cb S.I. (Ca:Cb) C. albicans >20000 1000 10000 400 <0.90 1:0.0400ATCC#10203 >20000 1000 10000 500 <1.00 1:0.0500 >20000 1000 10000 600<1.10 1:0.0600 >20000 1000 10000 800 <1.30 1:0.0800 >20000 1000 5000 400<0.65 1:0.0800 >20000 1000 5000 500 <0.75 1:0.1000 >20000 1000 5000 600<0.85 1:0.1200 >20000 1000 5000 800 <1.05 1:0.1600A: Surf. D

B: CTAC Media: 1/10 TSB

Inoculum size: 1.808E+05

PPM AI MIC Values (24 hrs) Test Alone Combination Ratio Organism CA CBCa Cb S.I. (Ca:Cb) S. aureus >20000 300 10000 80 <0.77 1:0.0080 ATCC#6538 >20000 300 10000 100 <0.83 1:0.0100 >20000 300 10000 200 <1.171:0.0200 >20000 300 5000 200 <0.92 1:0.0400 >20000 300 5000 80 <0.521:0.0160 >20000 300 5000 100 <0.58 1:0.0200 >20000 300 1750 80 <0.351:0.0457 >20000 300 1750 100 <0.42 1:0.0571 >20000 300 1750 200 <0.751:0.1143 >20000 300 875 200 <0.71 1:0.2286 >20000 300 437.5 200 <0.691:0.9143 >20000 300 218.75 200 <0.68 1:0.2286A: Surf. D

B: CTAC Media: 1/10 TSB

Inoculum size: 1.156E+06

P. aeruginosa ATCC#15442 No SynergyA: Surf. E

B: CTAC Media: PDB Inoculum 1.808E+05

A. niger ATCC 16404 No synergyA: Surf. E

B: CTAC Media: PDB Inoculum 1.808E+05

PPM AI MIC Values (48 hrs) Test Alone Combination Ratio Organism CA CBCa Cb S.I. (Ca:Cb) C. albicans >20000 2997 10000 1999 <1.17 1:0.1999ATCC 10203A: Surf. E

B: CTAC Media: 1/10 TSB

Inoculum size: 5.72E+07

P. aeruginosa ATCC#15442 No synergyA: Surf. E

B: CTAC Media: 1/10 TSB

Inoculum size: 1.8E+06

PPM AI MIC Values (24 hrs) Test Alone Combination Ratio Organism CA CBCa Cb S.I. (Ca:Cb) S. aureus >20000 200 10000 80 <0.90 1:0.0080 ATCC#6538 >20000 200 10000 100 <1.00 1:0.0100 >20000 200 5000 80 <0.651:0.0160 >20000 200 5000 100 <0.75 1:0.0200 >20000 200 2500 100 <0.631:0.0400 >20000 200 1750 100 <0.59 1:0.0571A: Surf. A

B: Tris Nitro

No synergy:S. aureus, A. niger, C. albicans, Ps. aeruginosaA: Surf. D

B: TrisNitro Media: 1/10 TSB

Inoculum size: 1.16E+08

PPM AI MIC Values (24 hrs) Test Alone Combination Ratio Organism CA CBCa Cb S.I. (Ca:Cb) Ps. aeruginosa >20000 800 20000 1000 <2.25 1:0.0500ATCC#15442 >20000 800 10000 1000 <1.75 1:0.1000 >20000 800 5000 1000<1.50 1:0.2000 >20000 800 2500 800 <1.13 1:0.3200 >20000 800 1750 1000<1.34 1:0.5714 >20000 800 875 800 <1.04 1:0.9143 >20000 800 437.5 600<0.77 1:1.3714 >20000 800 437.5 800 <1.02 1:1.8286 >20000 800 218.75 800<1.01 1:3.6571No synergy: S. aureus, A. niger, C. albicansA: Surf. E

B: TrisNitro Media: 1/10 TSB

Inoculum size: 5.727E+07 CFU/ml

PPM AI MIC Values (24 hrs) Test Alone Combination Ratio Organism CA CBCa Cb S.I. (Ca:Cb) Ps. aeruginosa >20000 1000 20000 1000 <2.00 1:0.0500ATCC#15442 >20000 1000 10000 1000 <1.50 1:0.1000 >20000 1000 5000 1000<1.25 1:0.2000 >20000 1000 2500 1000 <1.13 1:0.4000 >20000 1000 17501000 <1.09 1:0.5714 >20000 1000 875 1000 <1.04 1:1.1429 >20000 1000437.5 800 <0.82 1:1.8286 >20000 1000 437.5 1000 <1.02 1:2.2857 >200001000 218.75 800 <0.81 1:3.6571 >20000 1000 218.75 1000 <1.01 1:4.5714A: Surf. E

B: TrisNitro Media: 1/10 TSB Inoculumsize: 1.808E+06 CFU/ml

PPM AI MIC Values (24 hrs) Test Alone Combination Ratio Organism CA CBCa Cb S.I. (Ca:Cb) S. aureus >20000 500 20000 500 <2.00 1:0.0250 ATCC#6538 >20000 500 10000 400 <1.30 1:0.0400 >20000 500 5000 400 <1.051:0.0800 >20000 500 2500 400 <0.93 1:0.1600 >20000 500 1750 300 <0.691:0.1714 >20000 500 1750 400 <0.89 1:0.2286 >20000 500 875 400 <0.841:0.4571 >20000 500 437.5 400 <0.82 1:0.9143 >20000 500 218.75 400 <0.811:1.8286 >20000 500 218.75 500 <1.01 1:2.2857No synergy: A. niger, C. albicansA: Surf. A

B: DMO Media: PDB

Inoculum size: 1.156E+06 CFU/ml

PPM AI MIC Values (3^(rd) Day) Test Alone Combination Ratio Organism CACB Ca Cb S.I. (Ca:Cb) A. niger >20000 60 10000 50 <1.33 1:0.0050ATCC#16404 >20000 60 5000 50 <1.08 1:0.0100 >20000 60 2500 50 <0.961:0.0200 >20000 60 1750 50 <0.92 1:0.0286 >20000 60 875 50 <0.881:0.0571 >20000 60 437.5 50 <0.86 1:0.1143A: Surf. A

B: DMO Media: PDB

Inoculum size: 1.156E+06 CFU/ml

PPM AI MIC Values (3^(rd) Day) Test Alone Combination Ratio Organism CACB Ca Cb S.I. (Ca:Cb) C. albicans >20000 1000 10000 800 <1.30 1:0.0800ATCC#10231 >20000 1000 5000 800 <1.05 1:0.1600 >20000 1000 2500 800<0.93 1:0.3200 >20000 1000 875 800 <0.84 1:0.9143A: Surf. A

B: DMO Media: 1/10 TSB

Inoculum size: 8.134+07 CFU/ml

PPM AI MIC Values (24 hrs) Test Alone Combination Ratio Organism CA CBCa Cb S.I. (Ca:Cb) P. aeruginosa >20000 80 20000 60 <1.75 1:0.0030ATCC#15442A: Surf. A

B: DMO Media: 1/10 TSB

Inoculum size: 1.156E+06 CFU/ml

PPM AI MIC Values (24 hrs) Test Alone Combination Ratio Organism CA CBCa Cb S.I. (Ca:Cb) S. aureua >20000 50 20000 40 <1.80 1:0.0020 ATCC#6538A: Surf. D

B: DMO Media: 1/10 TSB

Inoculum size: 1.16E+08

P. aeruginosa ATCC#15442 No SynergyA: Surf. D

B: DMO Media: PDB

Inoculum size: 1.156E+06

PPM AI MIC Values (3rd day) Test Alone Combination Ratio Organism CA CBCa Cb S.I. (Ca:Cb) A. niger >20000 400 10000 300 <1.25 1:0.0300 ATCC#16404 >20000 400 5000 300 <1.0 1:0.0600 >20000 400 437.5 300 <0.771:0.6857A: Surf. D

B: DMO Media: PDB

Inoculum size: 5.726E

+05 CFU/ml

PPM AI MIC Values (48 hrs) Test Alone Combination Ratio Organism CA CBCa Cb S.I. (Ca:Cb) C. albicans >20000 1000 10000 800 <1.30 1:0.0800ATCC#10231 >20000 1000 5000 800 <1.05 1:0.1600A: Surf. D

B: 1135 Media: 1/10 TSB

Inoculum size: 1.16E+08

PPM AI MIC Values (24 hrs) Test Alone Combination Ratio Organism CA CBCa Cb S.I. (Ca:Cb) S. aureus >20000 80 10000 50 >1.13 1:0.0050ATCC#6538 >20000 80 10000 60 >1.25 1:0.0060 >20000 80 5000 50 <0.881:0.0100 >20000 80 5000 60 <1.00 1:0.0025 >20000 80 2500 60 <0.881:0.0120 >20000 80 1750 50 <0.71 1:0.0240 >20000 80 1750 60 <0.841:0.0286 >20000 80 875 50 <0.67 1:0.0343 >20000 80 875 60 <0.791:0.0571 >20000 80 437.5 50 <0.65 1:0.0686 >20000 80 437.5 60 <0.771:0.1143 >20000 80 218.75 60 <0.76 1:0.1371A: Surf. E

B: DMO Media: PDB

Inoculum size: 1808E+06

PPM AI MIC Values (48 hrs) Test Alone Combination Ratio Organism CA CBCa Cb S.I. (Ca:Cb) C. albicans >20000 1000 10000 600 <1.10 1:0.0600ATCC#10203 >20000 1000 10000 800 <1.30 1:0.0800 >20000 1000 5000 600<0.85 1:0.1200 >20000 1000 5000 800 <1.05 1:0.1600A: Surf. E

B: DMO Media: PDB

P. aeruginosa ATCC#15442 No SynergyA: Surf. E

B: DMO Media: PDB

Inoculum size: 1.56E+06 CFU/ml

PPM AI MIC Values (3^(rd) Day) Test Alone Combination Ratio Organism CACB Ca Cb S.I. (Ca:Cb) A. niger >20000 800 10000 600 <1.25 1:0.0600ATCC#16404 >20000 800 5000 600 <1.00 1:0.1200 >20000 800 437.5 600 <0.771:1.3714A: Surf. E

B: DMO Media: 1/10 TSB

Inoculum size: 1.80E+06 CFU/ml

PPM AI MIC Values (24 hrs) Test Alone Combination Ratio Organism CA CBCa Cb S.I. (Ca:Cb) ATCC# 6538 >20000 80 10000 50 <1.13 1:0.0050 >2000080 10000 60 <1.25 1:0.0060 >20000 80 5000 50 <0.88 1:0.0100 >20000 805000 60 <1.00 1:0.0120 >20000 80 2500 60 <0.88 1:0.0240 >20000 80 175060 <0.84 1:0.0343 >20000 80 875 60 <0.79 1:0.0686 >20000 80 437.5 50<0.65 1:0.1143 >20000 80 437.5 60 <0.77 1:0.1371 >20000 80 218.75 60<0.76 1:0.2743The following biocides had no synergy against any organism tested whenpaired with the following surfactants:Surf. A

Sodium Benzoate, TRIS NITRO

Surf. E

DMDMH

Surf. D

CS-1246, OPP, DMDMH

In the following combinations, the ratio of surfactant to biocide wheresynergy was observed were not commercially relevant, i.e., a ratio of1:0.2 or greater (less biocide relative to surfactant). At these ratios,the biocide levels in a formulated product would be too low to bepractical:Surf. A

DIDAC, IPBC

Surf. E

CMIT/MIT, IPBC, OIT, TTPC, WSCP

Surf. D

CMIT/MIT, OIT, DIDAC

(MBIT, IPBC, WSCP were synergistic only at 1:0.05 or worse except forone data point)

1. A synergistic microbicidal composition comprising: (a) a nonionicsurfactant with structure:R²O(CH₂CH(CH₃)O)₃(CH₂CH₂O)₇H where R² is a mixture of C₈-C₁₄ linearalkyl groups; and (b) tris(hydroxymethyl)nitromethane; wherein a weightratio of said nonionic surfactant to tris(hydroxymethyl)nitromethane isfrom 1:0.16 to 1:1.8286.
 2. The synergistic microbicidal composition ofclaim 1 in which said mixture of C₈-C₁₄ linear alkyl groups comprisesfrom 60 to 75 wt % C₈-C₁₀ linear alkyl groups and 25 to 40 wt % C₁₂-C₁₄linear alkyl groups.